1. Field of the Invention
Safe and stable packaging cell lines are described for preparing retroviral vectors. Also described are methods of production of therapeutics, vaccines for gene therapy of many genetic and acquired diseases (including AIDS and cancers).
2. Description of the Related Art
Retrovirus is employed as a common tool for stable gene transfer into human cells (See, e.g., Gilboa et al., BioTechiques (1986), 4(6): 504-512). The retroviral-mediated gene transfer system can be divided into two components (Miller and Rosman, BioTechniques (1989), 7:980-990). This design is based on the knowledge of studying murine retrovirus virus in the early 1980s (Joyner et al., Nature (1983), 305: 556-558; Mann et al., Cell (1983), 33: 153-159). The first component is the transfer vector itself, which generally include cis-acting sequences required for viral reverse transcription and integration and typically does not encode viral structure proteins. The second component is the retrovirus packaging construct or a cell line (helper cell line), which provides the viral proteins necessary for assembly of viral particles and transduction of recipient cells (Cone and Mulligan, Proc. Nat""l. Acad. Sci. USA (1984), 81: 6349-6353). A marker gene or therapeutic gene can be inserted into the transfer vector to be transferred into the recipient cells. Retroviral particles are produced by co-transfecting a transfer vector and a packaging construct into a cell line or by transfecting a transfer vector into a packaging cell line.
One of the potential safety concerns of using retrovirus in humans is the generation of replication-competent retrovirus (RCR) by homologous recombination between transfer vector and packaging construct during co-transfection. The RCR might cause unappreciated harms, such as T cell lymphoma, in the recipients (Danahue et al, J. Exp. Med. (1992), 176: 1125-1135). Several approaches have been developed to avoid the generation of RCR. One is to eliminate overlapping sequences between transfer vectors and packaging constructs. Another is to use a packaging cell line instead of co-transfection for viral particle production. Since only a single copy of the packaging sequence typically resides in the packaging cell line, instead of multiple copies in co-transfected cells, the chance to generate RCR is much reduced (Markowitz et al., J. Virol. (1988), 62(4): 1120-1124).
The retroviral vectors currently used in clinical gene therapy studies are derived from murine leukemia virus, which has an inherent property of infecting only proliferating cells (Miller et al., Molecular and Cellular Biology (1990), 10(8): 4239-42; Lewis and Emerman, J. Virology (1994), 68:510-516). Unfortunately, the majority of primary target cells for gene therapy trials are in quiescent stage, which results in low efficiency of gene transduction. Many protocols have been developed to enhance gene transfer efficiency, such as repeated transduction, growth factor stimulation, isolation of progenitor cells and use of high titer pseudotyped virus (Nolta et al., Exp. Hemotol. (1992 ), 20: 1065-1071; Van Beusechem et al., Gene Therapy (1995), 2: 245-255; Akina et al., J. Virology (1996), 70:22581-2585; Xu et al., Blood (1995) 86:141-146). However, the ex vivo manipulation of transduced cells causes the loss of pluripotentiality after engraftment into recipients (Van Beusechem et al., Gene Therapy (1995), 2: 245-255). An ideal vector for gene therapy would be able to directly deliver genes into primary cells in the recipients.
To approach this problem, HIV-1 has been developed as a viral vector for gene transfer in nondividing cells (Gallay et al., J. Virology (1996), 70: 1027-1032). Gene transfer has been demonstrated in quiescent cells using an HIV-1 based vector (Naldini et al., Science (1996), 272:263-267). In vitro, HIV-1 can infect primary cultures of monocyte-derived macrophages as well as cell cycle-arrested CD4+ T lymphoid cells (Strizki et al., J. Virology (1996), 70: 7654-62). Stable HIV-1 packaging cell lines have been developed to produce modest titer (103-5 cfu/ml) of virus (Corbeau et al., Proc. Nat""l. Acad. Sci. USA (1997), 93:14070-14075). However, the risk associated with the RCR virus evolved from co-transfection during HIV-1 vector preparation hinders its use as a gene transfer vector in healthy populations. Furthermore, there is no suitable animal model to demonstrate the safety of HIV-1 based vectors. To alleviate the safety concern of using HIV-1 vector for gene transfer in humans, lentiviral vectors derived from non-primate lentivirus has been described (Poeschla et al., Nature Medicine (1998), 4(3): 354-357; Olsen, Gene Therapy (1998), 5:1481-1487).
Simian Immunodeficiency Virus (SIV) exhibits cellular tropism similar to HIV-1, but it did not cause AIDS-like disease in humans who were accidentally infected with SIV (Rima et al., New England J. Medicine (1994), 330, 172-177-13; Khabbaz et al., Lancet (1992), 340:271-273). Recent reports suggested that SIV has broader co-receptor usage than HIV-1 (Chen et al., J. Virology (1997), 71: 2075-2714).
For the purpose of enhanced safety, the SIV genome was used to develop a gene transfer vector system for quiescent cells and to demonstrate the safety of using SIV-based gene transfer system in the primate model. Another potential usefulness of the SIV gene transfer vector is to deliver anti-HIV molecules for gene therapy of AIDS. In this case, the anti-HIV molecules (for example, and without limitation, anti-sense RNA and ribozymes) will not be self-inhibitory of the packaging systems. Described herein is a stable SIV packaging cell line, which can produce high-titer of viral particles. High viral titers can be produced even after pseudotyping with heterologous viral envelope protein(s), including, without limitation, glycoprotein G derived from Vesicular Stomatitis Virus. The pseudotyped SIV vectors can transduce primary cells including monocyte-derived macrophage and peripheral blood lymphocytes effectively.
Accordingly, described herein is a safe and stable gene transfer system for human gene therapy. A method also is provided for generating high-titer helper cell line and an assay for detecting replication competent virus in retroviral particle production. In another embodiment, a retroviral vector is provided that can inhibit HIV-1 replication.
A packaging nucleic acid, for instance a plasmid and derivatives thereof are provided, comprising SIV gag, pol and accessory genes (vif, tat, rev), with or without env gene, but lacking a functional SIV packaging sequence (an approximately 40 base pair sequence located between 5xe2x80x2 splicing donor site and initiation codon of gag). Also provided is a method for producing a stable packaging cell line by transferring the above-described packaging plasmid into a mammalian cell line, such as 293T or 293 cells.
A SIV transfer vector comprising cis-acting sequence for viral replication also is provided. One example includes the SIV 5xe2x80x2R and U5, about the first 320 base pair of the SIV gag coding sequence, the SIV rev-responding element (RRE) and the 3xe2x80x2 LTR.
A method for producing retroviral particles by transfecting the above-described SIV packaging cell line with transfer vector also is provided. If no envelope is provided in the packaging nucleic acid, an envelope gene expression plasmid is co-transfected with the transfer vector. The envelope gene expression plasmid includes an envelope gene that may be derived from any enveloped virus, which includes, but is not limited to, amphotropic murine leukemia virus, Sendai virus, hepatitis viruses and vesicular stomatitis virus.
A method for testing for RCR virus in cell culture and a monkey model is provided as described herein.
A recombinant SIV retroviral transfer vector encoding anti-sense or ribozyme sequence of HIV (anti-HIV molecules) also is provided and is useful for producing anti-HIV virus particles by the above-described method for producing retroviral particles. The use of retrovirus according to any of the preceding for the treatment of any genetic and acquired diseases, also is provided.
Retrovirus particles prepared according to any of the preceding also may be used to deliver an immunogen of any infectious disease and tumor to cells for vaccination purpose.